Cell Wall Assembly in Bacillus subtilis: Partial Conservation of Polar Wall Material and the Effect of Growth Conditions on the Pattern of Incorporation of New Material at the Polar Caps (original) (raw)
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Microbiology, 1989
Uranyl actetate staining of thin sections allowed a distinction to be made between cell wall material that contains teichoic acid and that which contains teichuronic acid. The stain was used to study the pattern of wall assembly in Bacillus subtilis undergoing transitions between growth conditions leading to incorporation of the different anionic polymers. The results showed that new material is incorporated along the inner surface of the cylindrical region of the wall confirming, by a more direct method, results obtained earlier with teichoic acid specific phages. New material appears to be evenly distributed along the inner surface and no evidence was obtained for the presence of specific zones of incorporation. Abbreviations: TA, teichoic acid; TU, teichuronic acid; TA or TU walls, cell wall material containing predominantly TA or TU as the anionic polymer, respectively.
Insertion andFateoftheCell WallinBacillus subtilis
1984
Cell wall assembly was studied in autolysin-deficient and-sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[ H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after'several generations. In the autolysin-deficient and chainforming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of at-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell Wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45°C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by' the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.
Applied and Environmental Microbiology, 2011
The surfaces of 8 bacterial and 23 archaeal species, including many hyperthermophilic Archaea, could be stained using succinimidyl esters of fluorescent dyes. This allowed us for the first time to analyze the mode of cell wall growth in Archaea by subculturing stained cells. The data obtained show that incorporation of new cell wall material in Archaea follows the pattern observed for Bacteria: in the coccoid species Pyrococcus furiosus incorporation was in the region of septum formation while for the rod-shaped species Methanopyrus kandleri and Methanothermus sociabilis, a diffuse incorporation of cell wall material over the cell length was observed. Cell surface appendages like fimbriae/pili, fibers, or flagella were detectable by fluorescence staining only in a very few cases although their presence was proven by electron microscopy. Our data in addition prove that Alexa Fluor dyes can be used for in situ analyses at temperatures up to 100°C.
Studies on the Bacterial Cell Wall XIII
Journal of Bacteriology, 1957
The chemical composition of the bacterial endospore has been investigated only in a few instances. In 1933, Virtanen and Pulkki studied the chemical composition of vegetative cells and spores of the same organism and concluded that there was no fundamental difference between the two in ash, water and protein contents. However, they pointed out the absence of enzymatic activities in the spore. Friedman and Henry
Control of Cell Shape in Bacteria
Cell, 2001
or intervene in cell shape determination is unknown Varley and Stewart, 1992). B. subtilis has two additional mreB-like genes, and disruption of one of these, mbl, also has an effect on cell shape (Abhayawardhane and South Parks Road Oxford OX1 3RE Stewart, 1995). At the primary sequence level, MreB and its relatives show weak similarity to the actin superfam-United Kingdom ily, and to another bacterial morphogenic protein, FtsA, which is required for cell division. However, the superfamily also contains a number of proteins of noncy-Summary toskeletal function, such as hexokinases and the chaperone Hsp70/DnaK (Bork et al., 1992).
Cell wall-polypeptide complexes in Bacillus subtilis
Carbohydrate Research, 1983
The cell surface of Bacillus subtilis contains several peptidoglycan-associated polypeptides. Cell walls were labeled with 1251 or %, and the products were digested with lysozyme. When the digests were chromatographed on Sephacryl S-200, peaks of radioactivity corresponding to molecular weights of 240,000,125,000, 20,000, 17,000, and 15,000 were observed. The walls solubilized by lysozyme were also subjected to sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis, and radioactive bands corresponding to apparent molecular weights of 24,000, 22,000, and 19,000 were found. Isoelectric focusing of the digests revealed the presence of a component having an isoelectric point of 3.7, and, possibly, of minor components having isoelectric points of 4.7 and 6.1. Proteases, including trypsin, subtilisin, and pronase, removed some of the radioactivity from [3"S]-labeled walls. Significant proportions of label from [35S]walls were solubilized by the peptide-bond-breaking agents cyanogen bromide and N-bromosuccinimide. Small proportions of radioactivity were released from labeled walls by hydroxylamine and trichloroacetic acid. Direct, amino acid analyses of the walls showed the presence of several amino acids not commonly regarded as constituents of peptidoglycan. Cell walls from a protease-deficient mutant, and from a wall preparation enriched in cell poles, contained similar proportions of amino acids. In addition. wall preparations from an autolysin-deficient mutant, and walls from protease hyper-producing strains, contained amino acids that could not be removed by rigorous extraction-procedures. The results suggest that the cell walls of Bacillus subtilis contain tightly, or covalently, bound protein molecules or polypeptides that are refractory to removal by denaturants.
Proceedings of the National Academy of Sciences, 2013
Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes. agrobacterium cell cycle | bacterial cell growth | bacterial cell division | peptidoglycan synthesis
Ultrastructure of a temperature-sensitive rod-mutant of Bacillus subtilis
Journal of …, 1970
Mutant 168ts-200B, resulting from nitrosoguanidine treatment of Bacillus subtilis 168 (trp-C2), exhibits a rod-to-sphere morphogenetic interconversion when the incubation temperature is 30 or 45 C, respectively. Ultrathin sections of rods grown at 30 C, after glutaraldehyde-osmium uranium-lead fixation and staining, show trilaminar cell walls with a well-developed underlying periplasm as in wild-type cells. However, the outer wall layer is irregular, and abnormal protrusions of wall material occur at the cross-walls. In contrast, cells growing at 45 C become rounded and are intersected randomly by irregular cross-walls which fail to split normally, resulting in large spherical masses. In these, the outer and inner wall layers and periplasm are lost, and the wall consists only of irregularly thickened and loosely organized middle layer. Wall ultrastructure is reversible in either direction as cell shape changes during temperature shifts. Mesosomes are rare and atypical at either temperature. It thus appears that cell wall ultrastructure is altered by the conditional (temperaturesensitive) mutation, and that loss of normal wall and submural organization is correlated with changes in cell size and shape as well as with inability to complete cell division. Preliminary studies after transformation of the mutant locus to another strain and growth at 45 C showed an increase in mucopeptide, loss of wall teichoic acid, failure of phage adsorption, and identical ultrastructural changes. The site of expression of the basic defect-be it in wall, submural region, or membrane-is undetermined. Conditional morphological mutants of Bacillus subtilis, as well as of Bacillus licheniformis, were reported by Rogers et al. (22). These rod mutants grew as irregular spheres on an inorganic salts-glucose-tryptophan medium lacking added sodium chloride, but grew as rods when sodium chloride or several amino acids were added (21). A morphologically similar variation, for which the condition is temperature instead of medium constituents, was recently described by some of us (2) in a mutant of B. subtilis 168 (trp-C2). In this report, we demonstrate changes in the ultrastructure of this mutant at the permissive and restrictive temperatures, and during transition from one temperature to the other. MATERIALS AND METHODS Organism. The temperature-sensitive rodmutant, designated 168ts-200B, was derived from B. subtilis 168 (trp-C2), designated 168ts+, as previously described (2). Culture. Media and growth conditions were described previously (2). Electron microscopy. At temperatures and times of incubation indicated, 2 ml of 5% glutaraldehyde, buffered to pH 7.2 in 0.2 M sodium cacodylate (24), was added to 25 ml of bacterial culture. The mixture was immediately centrifuged, and the cells were suspended in 3 ml of the buffered 5% glutaraldehyde for 1 hr at room temperature. A 0.3-ml amount of 1% tryptone medium (23) was then added, and the suspension was maintained at the same temperature for 1.5 hr. After centrifugation, the cells were placed in 0.2 M sucrose buffered at pH 7.2 in 0.1 M phosphate and held for 2 days at 4 C. Secondary fixation at room temperature in acetate-Veronal-buffered 1% osmium tetroxide, and subsequent washing, agar embedding, and treatment with uranyl acetate, were done according to Ryter and Kellenberger (23).
Proceedings of the National Academy of Sciences, 2015
Agrobacterium tumefaciens elongates by addition of peptidoglycan (PG) only at the pole created by cell division, the growth pole, whereas the opposite pole, the old pole, is inactive for PG synthesis. How Agrobacterium assigns and maintains pole asymmetry is not understood. Here, we investigated whether polar growth is correlated with novel pole-specific localization of proteins implicated in a variety of growth and cell division pathways. The cell cycle of A. tumefaciens was monitored by time-lapse and superresolution microscopy to image the localization of A. tumefaciens homologs of proteins involved in cell division, PG synthesis and pole identity. FtsZ and FtsA accumulate at the growth pole during elongation, and improved imaging reveals FtsZ disappears from the growth pole and accumulates at the midcell before FtsA. The L,D-transpeptidase Atu0845 was detected mainly at the growth pole. A. tumefaciens specific pole-organizing protein (Pop) PopZAt and polar organelle development ...